Chemical modification of papain. I. Reaction with the chloromethyl ketones of phenylalanine and lysine and with phenylmethyl-sulfonyl fluoride.
Chemical modification of papain. I. Reaction with the chloromethyl ketones of phenylalanine and lysine and with phenylmethyl-sulfonyl fluoride.
复制标题
木瓜蛋白酶的化学修饰。
DOI：
10.1016/0003-9861(68)90304-4
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发表时间：
1968
影响因子：
3.9
通讯作者：
J. Perez
中科院分区：
文献类型：
--
作者：
J. Whitaker;J. Perez
木瓜蛋白酶与甲苯磺酰基-l-苯丙氨酸和甲苯磺酰基-l-赖氨酸的氯甲基酮的反应是快速的，并且导致活性完全丧失。氯甲基酮与木瓜蛋白酶的唯一巯基（在半胱氨酸残基25号上）特异性反应，如巯基分析、氨基酸分析、分光光度数据所示，并且不与木瓜汞蛋白酶反应。α-N-对甲苯磺酰基-L-苯丙氨酸氯甲基酮与木瓜蛋白酶反应的二级反应速率常数在0 °时为200 m − 1 sec − 1，并且取决于pK8.90的单一亲质子基团。反应速率随着pH值的增加而增加，这表明反应性物质是硫醇阴离子。苯甲基磺酰氟在pH 7和0 °下迅速抑制木瓜蛋白酶。失活的酶可以通过用二硫苏糖醇处理而完全再活化。在与苯甲基磺酰氟反应后制备羟基木瓜蛋白酶的尝试尚未成功。
Reaction of papain with the chloromethyl ketones of tosyl-l-phenylalanine and tosyl-l-lysine is rapid and results in a complete loss of activity. The chloromethyl ketones react specifically with the sole sulfhydryl group (on cysteine residue No. 25) of papain as shown by sulfhydryl group analysis, amino acid analysis, spectrophotometric data, and failure to react with mercuripapain. The limiting second-order reaction rate constant for reaction of α-N-tosyl-l-phenylalanine chloromethyl ketone with papain was found to be 200m−1sec−1at 0 ° and to be dependent upon a single prototropic group of pK8.90. The rate of the reaction increases with increasing pH, an indication that the reactive species is the thiol anion. Phenylmethylsulfonyl fluoride inhibits papain rapidly at pH 7 and 0 °. The inactive enzyme can be fully reactivated by treatment with dithiothreitol. Attempts to prepare hydroxypapain after reaction with phenylmethylsulfonyl fluoride have not been successful.